MOLECULAR AND CELLULAR BIOLOGY, May 1989, p. 2050-2057 Vol. 9, No. 5 0270-7306/89/052050-08$02.00/0 Copyright © 1989, American Society for Microbiology The Two Positively Acting Regulatory Proteins PH02 and PH04 Physically Interact with PHOS Upstream Activation Regions KURT VOGEL,1 WOLFRAM HORZ,2 AND ALBERT HINNENl* Biotechnology Department, CIBA-GEIGY AG, CH4002 Basel, Switzerland,' and Institut fur Physiologische Chemie, Physikalische Biochemie, und Zellbiologie der Universitdt Munchen, 8000 Munich 2, Federal Republic ofGermany Received 12 September 1988/Accepted 7 February 1989 The repressible acid phosphatase gene PHOS of Saccharomyces cerevisiae requires the two positively acting regulatory proteins PHO2 and PHO4 for expression. pho2 or pho4 mutants are not able to derepress the PHOS gene under low-Pi conditions. Here we show that both PHO2 and PHO4 bind specifically to the PHOS promoter in vitro. Gel retardation assays using promoter deletions revealed two regions involved in PH04 binding. Further characterization by DNase I footprinting showed two protected areas, one located at -347 to -373 (relative to the ATG initiator codon) (UASpl) and the other located at -239 to -262 (UASp2). Exonuclease m footprint experiments revealed stops at -349 and -368 (UASpl) as well as at -245 and -260 (UASp2). Gel retardation assays with the PH02 protein revealed a binding region that lay between the two PH04-binding sites. DNase I footprint analysis suggested a PH02-binding site covering the region between -277 and -296. The promoter region of the strongly regulated acid phos- press). Interestingly, the pho2 mutation showed no influence phatase gene PH05 (4) has been recently analyzed by on the basal level of TRP4 expression but rather modulated deletion mapping (6, 19, 24). In addition to the TATA box, the general control response of TRP4. two regions were found (24) to be essential for activation of the PHOS promoter. Sequence comparisons defined four MATERIALS AND METHODS elements from which a 19-base-pair dyad consensus se- quence was deduced. Three of the elements, called UASp, Escherichia coli vector for PH04 and PH02 expression. The were located within deletions showing reduced transcription PH04 gene (13, 15) was reisolated from a S288C background of PHOS (24), at -367, -245, and -185 relative to the by screening an ordered centromere library (25). An NcoI translational start. site at the ATG of PH04 was introduced by site-specific Not much is known about the physical interaction of mutagenesis (14). The PH04 gene (NcoI-BamHI fragment) regulatory proteins with the PHOS promoter. Since muta- was placed under control of the inducible PL promoter by tions defective in PH02 or PH04 are epistatic to all other using the vector pPLmu, a derivative of PLc24 (23) contain- regulatory mutations and show a negative phenotype, it is ing the phage Mu Ner gene ribosome-binding site with an likely that the PHO2 and PHO4 proteins are positive tran- NcoI site at its ATG start codon (obtained from Biogen). scription factors that interact with the PHOS promoter The PH02 gene (25) was fused to the same vector but DNA. The finding that a pho2 mutation can be comple- carrying a different ribosome-binding site (no. 27-4878-01 mented by overexpression of the PHO4 protein (20) is a and 27-4898-01; Pharmacia, Uppsala, Sweden; vector kindly further indication that the PHO2 and PHO4 proteins exert provided by A. Schmitz). The correct fusion of this ribo- their functions at the same late hierarchical level of PH05 some-binding site to the PH02 gene was achieved with an activation. adapter oligonucleotide that fits to the BamHI site of the Extensive studies of the chromatin fine structure of the ribosome-binding site and the EcoRI site of the PH02 gene. PH05 gene (1, 2, 5, 7) have been done. Interestingly, the The correct in-frame fusions of both constructions were element at -367, which is essential for gene induction (24), verified by sequencing the junctions. resides in the center of a hypersensitive region under condi- Partial purification of E. coli-expressed PH04 and PH02 tions of PH05 repression (1, 2). proteins. The E. coli expression vector with the PH04 or In this paper, we show that the two positive regulatory PH02 genes was transformed into JM109 cells carrying the proteins PHO4 and PHO2 bind specifically to PH05 pro- temperature-sensitive X repressor pcI857 on a plasmid. moter DNA. Footprint analysis revealed two regions Upon heat induction, heterologous protein accumulated to a (UASp1 and UASp2) protected by the PHO4 protein. The few percent of the level of total cellular protein. For binding PH05 promoter was found to have a PHO2-binding site studies, a soluble cell lysate (1.2 g of total protein for PHO4 located between the two PHO4-binding sites. These results and 0.5 g for PHO2) was partially purified by applying it to a are in agreement with data obtained by our fine-structure DE-52 column (30-ml bed volume) in 100 mM Tris (pH deletion mapping (24) but differ to some extent from data 7.5)-0.1 mM EDTA-1 mM phenylmethylsulfonyl fluoride published by Nakao et al. (19) and Bergman et al. (6). and rinsing the preparation with the same buffer. The Recently, it was shown that BAS2 (allelic to PHO2) also flowthrough fractions were passed through an Affi-Gel- binds to the HIS4 promoter region (3) and regulates the basal heparin (Bio-Rad Laboratories, Richmond, Calif.) column level (30-ml bed volume). Protein was eluted from the heparin- of HIS4 expression. Furthermore, PHO2 was found to sepharose column by applying a Tris gradient. The pooled bind very strongly to the TRP4 promoter (G. Braus, H.-U. fractions contained approximately l0o PH04 and 2% Mosch, K. Vogel, A. Hinnen, and R. Hutter, EMBO J., in PHO2, respectively. Gel retardation assays. For gel retardation assays (10), the * Corresponding author. recessed 3' ends of the DNA fragments were filled in with 2050 VOL. 9, 1989 INTERACTIONS OF PHO2 AND PHO4 WITH THE PHOS GENE 2051 TATA -*Si//ATG +1 -541 -469 - 367 -245 -185 -100 ItI B B A H A -O-400 32 (1) a 11 -, -36 (1w ) A12 -39-346 (0.1) A13 -350432 (0.6) 3.4 (0.4) 15- -2n (o.9) 16 -283-262 (l) A 17 -255 174 (0.2) A A8-22618 i2 -174, ( l) FIG. 1. The PHOS promoter. Negative numbers indicate elements found by deletion mapping (24). Listed beneath the sequence are some of the deletions used. Numbers in brackets show activities of the deletions in vivo. Restriction sites: A, AatII; B, BamHI; BE, BstEII; C, ClaI; H, HinPI; M, MnlI; S, Sail. Klenow polymerase as described by Maniatis et al. (17), counts) was added to 38 ,ul (final volume) of reaction mixture using a-35S-labeled deoxynucleoside triphosphates. Approx- containing 50 mM NaCl, 10 mM Tris (pH 7.5), 2 mM imately 1 ng of labeled fragments was added to a 20-,ul (final dithiothreitol, 5 mM MgCl2, 0.1 mM EDTA, and 3 ,ug of volume) reaction mixture containing 100 mM NaCl, 10 mM double-stranded poly(dI-dC) competitor DNA. Then 1 ,ug of Tris (pH 7.5), 2 mM dithiothreitol, 0.1 mM EDTA, and 2 ,ug partially purified PHO4 protein was added. After the binding of double-stranded poly(dI-dC) competitor DNA. Finally, reaction was allowed to proceed for 15 min at 25°C, 2 RI (12.5 0.2 ,ug of PHO4 or 2 ,ug of PHO2 protein (partially purified) U) of exonuclease III (Boehringer Mannheim Biochemicals, was added, and the binding reaction was allowed to proceed Indianapolis, Ind.) was added for 3, 6, or 12 min at 25°C. The for 15 min at 25°C. The samples were loaded immediately reaction was stopped by addition of 4.8 of 500 mM Tris (under voltage) onto a 6% polyacrylamide gel (acrylamide- (pH 8.0)-S50 mM EDTA-5% sodium dodecyl sulfate and 2 bisacrylamide weight ratio of 30:1) made in 0.5x TBE. (50 of proteinase K (10 mg/ml). After proteinase K treatment, mM Tris [pH 8.3], 50 mM boric acid, and 1.25 mM EDTA). the samples were loaded onto a standard sequencing gel. Electrophoresis was performed at 350 V for 2 to 3 h at room temperature. The dried gels were exposed to sensitive X-ray RESULTS films (Eastman Kodak Co., Rochester, N.Y.). DNase I footprinting with PHO4. DNase I footprint assays PH04 and PH02 proteins bind specificafly to the PHOS were adapted from the method of Galas and Schmitz (9). A promoter. The promoter region of PH05 was screened for 32P-end-labeled DNA fragment (approximately 20,000 functionally important DNA sequences by the construction Cerenkov counts) was added to 20 ,ul (final volume) of of a series of deletion mutants (24) (Fig. 1). To determine reaction mixture containing 50 mM NaCl, 10 mM Tris (pH whether the regulatory proteins PHO2 or PHO4 bound 7.5), 2 mM dithiothreitol, 1 mM EDTA, 0.5 mM MgCl2, and directly to the PHOS promoter, we decided to analyze this 1.5 ,ug of poly(dI-dC) competitor DNA. After addition of 0.5 possible protein-DNA interaction in vitro. Both proteins ,ug of partially purified PHO4 protein, the binding reaction were expressed in E. coli (see Materials and Methods), was allowed to proceed for 15 min at 25°C. The samples were which eliminated the possibility of contamination by other transferred on ice, and 2 ,ul of DNase I (50 p.g/ml) freshly yeast proteins. After partial purification of the expressed diluted in 10 mM Tris (pH 7.5)-25 mM CaCl2 was added to proteins, gel retardation experiments were performed with the reaction mixtures.